Purification of rapamycin derivatives using temperature induced phase separation

ABSTRACT

The present invention provides methods for obtaining purified rapamycin derivatives, including purified Biolimus A9. A crystalline form of Biolimus A9 is also described.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application is a continuation of International ApplicationNo. PCT/US2014/030602, filed Mar. 17, 2014, which claims priority toU.S. Provisional Patent Application No. 61/799,857, filed Mar. 15, 2013,the disclosures of which are incorporated herein by reference in theirentirety.

BACKGROUND OF THE INVENTION

This invention provides for an economic and effective means to purify40-O-rapamycin derivatives from unwanted byproducts having similarpolarity generated during synthesis. Rapamycin is also known assirolimus, CAS [53123-88-9]). It is a commercially available macrolidenatural product synthesized by Streptomyces hygroscopicus. A preferredderivative, 40-O-(2-ethoxyethyl) rapamycin, Umirolimus (INN/USAN),Biolimus A9™ (also known as BA9™) is an active pharmaceutical ingredientdeveloped as a drug coating for coronary stents to prevent smooth musclecell proliferation and restenosis. Other members of this ‘limus’ familyinclude everolimus (CAS [159351-69-6]), zotarolimus (CAS [221877-54-9])and temsirolimus (CAS [162635-04-03]). Members of the family are knownto possess immunosuppressive, antifungal, anti-tumor, and/oranti-inflammatory activity in vivo and are useful in the treatment oftransplantation rejection, infectious diseases, autoimmune diseases, andconditions characterized by excessive cell proliferation.

The chemical structure of BA9 consists of a 31-membered triene macrolidelactone that preserves the core rapamycin ring structure and differsonly in the addition of a side chain at position 40 in which thehydroxyl group of rapamycin has been alkylated with an ethoxyethylgroup.

The chemical structure of BA9 compared to sirolimus and other sirolimusderivatives is provided in FIG. 1. BA9 is structurally related torapamycin (also known as sirolimus). The structure consists of therapamycin 31-membered macrolide triene lactone ring withethoxyethylation at position 40.

BA9, like sirolimus, binds to the intracellular immunophilin proteinFKBP12. It is believed that the resulting macrolide/FKBP12 complex thenbinds, in a manner similar to sirolimus, to mTOR, a protein critical forcell cycle progression. Inactivation of mTOR results in suppression ofseveral specific signal transduction pathways and arrest of the cellcycle at the G1 to S phase.

Given the therapeutic value of BA9 and other rapamycin derivatives,improved processes for preparation of this family of active agents isdesired. The present invention addresses this and other needs.

BRIEF SUMMARY OF THE INVENTION

The present invention provides method for obtaining a purified, solidcompound having a structure according to Formula I:

wherein R¹ is selected from the group consisting of H;R^(a)—(O)_(d)—R^(b), wherein R^(a) is C₁₋₅alkylene, R^(b) is C₁₋₅alkylor C₁₋₅alkylene-OH, and the subscript d is an integer selected from 0-1;C₁₋₅alkyl; C₆₋₁₀arylC₁₋₅alkyl; hydroxyC₁₋₅alkyl; C₆₋₁₀arylC₁₋₅alkoxy;C₁₋₅alkoxyC₁₋₅alkyl; acyl; acylC₁₋₅alkyl; aminoC₁₋₅alkyl;C₁₋₅alkylaminoC₁₋₅alkyl; acylaminoC₁₋₅alkyl;C₁₋₅alkoxycarbonylaminoC₁₋₅alkyl; and C₆₋₁₀aryl.

The method includes: a) forming a mixture comprising a crude compoundhaving a structure according to Formula I and a non-polar organicsolvent under conditions sufficient to dissolve the compound; b)solidifying at least a portion of the compound having the structureaccording to Formula I; c) separating at least a portion of thesolidified compound from the solvent in the mixture; and therebyobtaining the purified, solid compound.

In another embodiment this invention provides for a crystallized form ofBA9.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the chemical structures of sirolimus, Biolimus A9, andrelated derivatives.

FIG. 2 shows a scheme for the preparation of rapamycin derivatives andpurification of the derivatives according to the methods of theinvention.

FIG. 3 shows an X-ray powder diffraction (XRPD) pattern observed for BA9Lot 2A.

FIG. 4 shows a differential scanning calorimetry (DSC) thermogramobserved for BA9 Lot 2A.

FIG. 5 shows an XRPD pattern observed for BA9 Lot 2B.

FIG. 6 shows a DSC thermogram observed for BA9 Lot 2B.

FIG. 7 shows an optical micrograph observed for BA9 Lot 2B.

FIG. 8 shows a dynamic vapor sorption (DVS) kinetic plot observed forBA9 Lot 2B.

FIG. 9 shows a XRPD pattern overlay observed for BA9 Lot 2B after a DVSexperiment and a reference pattern of BA9 Form I.

FIG. 10 shows and overlay of the XRPD patterns obtained from solventslurries and a reference pattern of BA9 Form I.

FIG. 11 shows an overlay of the XRPD patterns obtained in anti-solventcrystallization experiments and a reference pattern of BA9 Form I.

FIG. 12 shows an infrared spectrum of BA9 Form I, obtained using asample in a KBr pellet.

DETAILED DESCRIPTION OF THE INVENTION I. General

The present invention provides improved methods for purifying rapamycinderivatives including Biolimus A9 (BA9). The process includessolidification of a rapamycin derivative from non-polar organicsolvents, followed by isolation of the purified product and optionalprecipitation from a mixture of a polar organic solvent and water. Thevarious steps of the process are described herein. Effective removal ofvarious impurities was discovered to result from the surprisinglysimple, economical process. These advantages are described in detailbelow.

The discovery that a simple solidification by lowering temperature wouldseparate the 40-O derivatives so cleanly from unwanted 40-O derivativeimpurities and non-40-O derivatives was surprising and unpredictable. Itis economically advantageous because the unwanted by-products are oftentimes of a similar polarity to the desired product and they aredifficult to separate using large scale chromatography techniques. Themethods can be used for preparation of a crystalline form of BA9 thatdemonstrates superior stability.

II. Definitions

The term “purified” refers to a compound that has been processed toremove impurities. Impurities can include solvents, reagents used toprepare the compound, starting materials, and byproducts of a reactiongiving rise to the compound. In some embodiments, a purified compound issubstantially free of other species.

The term “crude compound” refers to a mixture containing a desiredcompound (such as a compound of Formula I as described herein) and atleast one other species selected from a solvent, a reagent such as abase, a starting material, and a byproduct of a reaction giving rise tothe desired compound.

“Alkyl” refers to a straight or branched, saturated, aliphatic radicalhaving the number of carbon atoms indicated. Alkyl can include anynumber of carbons, such as C₁₋₂, C₁₋₃, C₁₋₄, C₁₋₅, C₁₋₆, C₁₋₇, C₁₋₈,C₁₋₉, C₁₋₁₀, C₂₋₃, C₂₋₄, C₂₋₅, C₂₋₆, C₃₋₄, C₃₋₅, C₃₋₆, C₄₋₅, C₄₋₆ andC₅₋₆. For example, C₁₋₆ alkyl includes, but is not limited to, methyl,ethyl, propyl, isopropyl, butyl, isobutyl, sec.butyl, tert.butyl,pentyl, isopentyl, hexyl, etc. Alkyl can also refer to alkyl groupshaving up to 20 carbons atoms, such as, but not limited to heptyl,octyl, nonyl, decyl, etc.

“Alkylene” refers to a straight or branched, saturated, aliphaticradical having the number of carbon atoms indicated, and linking atleast two other groups, i.e., a divalent hydrocarbon radical. The twomoieties linked to the alkylene can be linked to the same atom ordifferent atoms of the alkylene group. For instance, a straight chainalkylene can be the bivalent radical of —(CH₂)_(n)—, where n is 1, 2, 3,4, 5 or 6. Representative alkylene groups include, but are not limitedto, methylene, ethylene, propylene, isopropylene, butylene, isobutylene,sec-butylene, pentylene and hexylene.

“Alkenyl” refers to a straight chain or branched hydrocarbon having atleast 2 carbon atoms and at least one double bond. Alkenyl can includeany number of carbons, such as C₂, C₂₋₃, C₂₋₄, C₂₋₅, C₂₋₆, C₂₋₇, C₂₋₈,C₂₋₉, C₂₋₁₀, C₃, C₃₋₄, C₃₋₅, C₃₋₆, C₄, C₄₋₅, C₄₋₆, C₅, C₅₋₆, and C₆.Alkenyl groups can have any suitable number of double bonds, including,but not limited to, 1, 2, 3, 4, 5 or more. Examples of alkenyl groupsinclude, but are not limited to, vinyl (ethenyl), propenyl, isopropenyl,1-butenyl, 2-butenyl, isobutenyl, butadienyl, 1-pentenyl, 2-pentenyl,isopentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl,3-hexenyl, 1,3-hexadienyl, 1,4-hexadienyl, 1,5-hexadienyl,2,4-hexadienyl, or 1,3,5-hexatrienyl.

“Alkynyl” refers to either a straight chain or branched hydrocarbonhaving at least 2 carbon atoms and at least one triple bond. Alkynyl caninclude any number of carbons, such as C₂, C₂₋₃, C₂₋₄, C₂₋₅, C₂₋₆, C₂₋₇,C₂₋₈, C₂₋₉, C₂₋₁₀, C₃, C₃₋₄, C₃₋₅, C₃₋₆, C₄, C₄₋₅, C₄₋₆, C₅, C₅₋₆, andC₆. Examples of alkynyl groups include, but are not limited to,acetylenyl, propynyl, 1-butynyl, 2-butynyl, isobutynyl, sec-butynyl,butadiynyl, 1-pentynyl, 2-pentynyl, isopentynyl, 1,3-pentadiynyl,1,4-pentadiynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 1,3-hexadiynyl,1,4-hexadiynyl, 1,5-hexadiynyl, 2,4-hexadiynyl, or 1,3,5-hexatriynyl.

“Aryl” refers to an aromatic ring system having any suitable number ofring atoms and any suitable number of rings. Aryl groups can include anysuitable number of ring atoms, such as, 6, 7, 8, 9, 10, 11, 12, 13, 14,15 or 16 ring atoms, as well as from 6 to 10, 6 to 12, or 6 to 14 ringmembers. Aryl groups can be monocyclic, fused to form bicyclic ortricyclic groups, or linked by a bond to form a biaryl group.Representative aryl groups include phenyl, naphthyl and biphenyl. Otheraryl groups include benzyl, having a methylene linking group. Some arylgroups have from 6 to 12 ring members, such as phenyl, naphthyl orbiphenyl. Other aryl groups have from 6 to 10 ring members, such asphenyl or naphthyl. Some other aryl groups have 6 ring members, such asphenyl.

“Alkoxy” refers to an alkyl group having an oxygen atom that connectsthe alkyl group to the point of attachment: alkyl-O—. As for alkylgroup, alkoxy groups can have any suitable number of carbon atoms, suchas C₁₋₆. Alkoxy groups include, for example, methoxy, ethoxy, propoxy,iso-propoxy, butoxy, 2-butoxy, iso-butoxy, sec-butoxy, tert-butoxy,pentoxy, hexoxy, etc.

“Carbonyl” refers to a moiety consisting of a carbon-oxygen double bond(i.e., —C(O)—).

“Acyl” refers to a moiety including a carbonyl group, as describedherein, bound to an alkyl group, and alkenyl group, or an alkynyl group,as described herein.

“Forming a mixture” and “contacting” refers to the process of bringinginto contact at least two distinct species such that they mix together.

“Non-polar organic solvent” refers to a carbon-based substance that isliquid at or near room temperature, substantially free of water, andcharacterized by a low dielectric constant (i.e., less than about 5).Examples of non-polar organic solvents that are suitable for use hereininclude, but are not limited to, hexane, heptane, ligroin, cyclohexane,pentane, n-octane, iso-octane, methylcyclohexane, mineral oil, diethylether, diisopropyl ether, methyl t-butyl ether, 1,4-dioxane, chloroform,aromatic hydrocarbon solvents (such as benzene and toluene),cyclopentane, n-octane, iso-octane and methylcyclohexane, for example.

“Alkane organic solvent” refers to a saturated hydrocarbon that isliquid at or near room temperature and substantially free of water.Examples of alkane organic solvents include hexane, heptane, ligroin,cyclohexane, pentane, n-octane, iso-octane, methylcyclohexane, andmineral oil.

“Polar organic solvent” refers to a carbon-based substance that isliquid at or near room temperature, substantially free of water, andcharacterized by a moderate to high dielectric constant (i.e., greaterthan about 5). Examples of polar organic solvents includedimethylformamide, dimethyl sulfoxide, propylene carbonate,acetonitrile, methanol, ethanol, isopropanol, t-butanol,tetrahydrofuran, and acetone.

“Solubilizing” refers to the process of dissolving a solid form of asubstance in a solvent to form a solution. The entirety of a solidsubstance, or any fraction thereof, can be caused to dissolve.Undissolved material can be present in the solvent in the form of asuspension

“Cooling” refers to the process of reducing the temperature of asubstance or mixture of substances.

“Solidifying” refers to the process of causing a compound in a solutionto coalesce into a solid form of the substance. The entirety of acompound in a solution, or any fraction thereof, can be caused tosolidify. The solid form can be an amorphous or crystalline substance.“Precipitating” refers to solidifying a substance in an amorphous form.

“Solid” refers to a condensed form of a compound that is not a gas,liquid, or solution. A solid can include one species or a mixture of twoor more species. A solid compound can be a crystalline form, anamorphous form, a glass, a foam, or a mixture of two or more forms.

“Crystalline form” refers to a solid form of a compound wherein theconstituent molecules are packed in a regularly ordered, repeatingpattern. A crystalline form can be triclinic, monoclinic, orthorhombic,tetragonal, trigonal, hexagonal, or cubic. A crystalline form cancontain one or more regions, i.e., grains, with distinct crystalboundaries. A crystalline solid can contain two or more crystalgeometries.

“Amorphous form” refers to a solid form of a compound having no definitecrystal structure, i.e., lacking a regularly ordered, repeating patternof constituent molecules.

“Unsolidified” refers to a compound that is not in a solid form. Anunsolidified compound can be, for example, dissolved in a solution orsuspended in a colloid.

“Separating” refers to the process of isolating at least a portion of acompound from a mixture containing the compound and at least one othersubstance. The isolated compound is substantially free at least one ofthe other substances present in the mixture.

“Reflux” refers to the process of boiling of a solvent while condensingthe solvent vapors and returned the condensed solvent to the boilingpot. Reflux is generally conducted at or near the boiling point of asolvent or mixture of solvents at a particular pressure.

“Drying” refers to the removal of a liquid species, such as a solvent,from a compound. Drying is generally conducted by heating the compound,reducing the pressure under which the compound is stored, or both.

The terms “about” and “around,” as used herein to modify a numericalvalue, indicate a close range surrounding that explicit value. If “X”were the value, “about X” or “around X” would indicate a value from 0.9Xto 1.1X, and more preferably, a value from 0.95X to 1.05X. Any referenceto “about X” or “around X” specifically indicates at least the values X,0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, and1.05X. Thus, “about X” and “around X” are intended to teach and providewritten description support for a claim limitation of, e.g., “0.98X.”

III. Embodiments of the Invention

The present invention provides method for obtaining a purified, solidcompound having a structure according to Formula I:

wherein R¹ is selected from the group consisting of H;R^(a)—(O)_(d)—R^(b), wherein R^(a) is C₁₋₅alkylene, R^(b) is C₁₋₅alkylor C₁₋₅alkylene-OH, and the subscript d is an integer selected from 0-1;C₁₋₅alkyl; C₆₋₁₀arylC₁₋₅alkyl; hydroxyC₁₋₅alkyl; C₆₋₁₀arylC₁₋₅alkoxy;C₁₋₅alkoxyC₁₋₅alkyl; acyl; acylC₁₋₅alkyl; aminoC₁₋₅alkyl;C₁₋₅alkylaminoC₁₋₅alkyl; acylaminoC₁₋₅alkyl;C₁₋₅alkoxycarbonylaminoC₁₋₅alkyl; and C₆₋₁₀aryl.

The method includes: a) forming a mixture comprising a crude compoundhaving a structure according to Formula I and a non-polar organicsolvent under conditions sufficient to dissolve the compound; b)solidifying at least a portion of the compound having the structureaccording to Formula I; c) separating at least a portion of thesolidified compound from the solvent in the mixture; and therebyobtaining the purified, solid compound.

In some embodiments, the crude compounds used in the methods of theinvention are prepared according to the process shown in FIG. 2. Theprocess includes reaction of rapamycin with a suitable triflate at acontrolled temperature, followed by work-up and optional isolation ofthe products. Isotopically labeled starting materials, such asdeuterated materials, can be used to prepare isotopically labeledrapamycin derivatives. The methods of the invention generally includeseparating a compound of Formula I from a reaction mixture includingsolvents, reagents including bases, and starting materials according toFormula II and Formula III (see FIG. 2). Suitable separation techniquesinclude, but are not limited to, filtration of a solidified compound ofFormula I, centrifugation of a solidified compound of Formula I,distillation, liquid extraction, sublimation, and chromatographictechniques. Examples of chromatographic techniques include, but are notlimited to, normal-phase column chromatography (i.e., silica gel columnchromatography), reverse-phase column chromatography, and thin-layerchromatography. Two or more separation techniques can be conducted incombination to separate the compound of Formula I. In some embodiments,the chromatography is silica gel chromatography with hexane or heptaneand ethyl acetate.

Chromatography and other separation techniques provide crude compoundsof Formula I with varying levels of purity. The crude compounds containa compound of Formula I, as well as at least one other species selectedfrom a solvent, a reagent such as a base, a starting material accordingto Formula II or Formula III, and byproduct(s) of the rapamycinderivatization reaction. In general, a crude compound contains at least40% of a compound of Formula I by weight. In certain embodiments, acrude compound contains at least 50% of a compound of Formula I byweight. The crude compound can include, for example, from about 40% toabout 99%, or from about 50% to about 99%, or from about 75% to about95%, of a compound of Formula I by weight. In some embodiments, thecrude compound contains from about 90% to about 95% of a compound ofFormula I by weight.

Any suitable solvent can be used in the methods of the invention. Ingeneral, suitable solvents are non-polar. Preferred solvents includealkane organic solvents (i.e., saturated hydrocarbon solvents) andalkene organic solvents. Examples of alkane organic solvents include,but are not limited to, hexane, heptane, ligroin (i.e. petroleum ether),cyclohexane, octane, pentane, and mineral oil (including paraffinic oilsand naphthenic oils). In some embodiments, the non-polar organic solventis selected from hexane, heptane, ligroin, cyclohexane, pentane,n-octane, iso-octane, methylcyclohexane, mineral oil, diethyl ether,diisopropyl ether, methyl t-butyl ether, 1,4-dioxane, chloroform,benzene, toluene, cyclopentane, n-octane, iso-octane andmethylcyclohexane, and mixtures thereof. In some embodiments, thenon-polar organic solvent is selected from hexane, heptane, ligroin,octane, cyclohexane and mixtures thereof. In some embodiments, thenon-polar organic solvent is hexane. In some embodiments, the non-polarorganic solvent is heptane. Other non-polar solvents can be useful inthe methods of the invention depending on the properties of theparticular rapamycin derivative.

Any suitable quantity of solvent can be used in the methods of theinvention. In general, the amount of solvent used is sufficient todissolve the compound of Formula I. Typically, the amount of solventranges from about 1 part to about 500 parts, by weight, per part crudecompound. The solvent:crude ratio can be from about 1:1 to about 500:1,or from about 50:1 to about 250:1 by weight. The solvent:crude ratio canbe about 95:1, or about 100:1, or about 105:1, or about 110:1, or about115:1, or about 120:1 by weight. In some embodiments, the solvent:cruderatio is about 110:1 by weight. The crude weight in the solvent:cruderatio can refer to the total weight of the crude mixture or to theweight of the compound of Formula I in the mixture as determined, forexample, by HPLC or another analytical technique.

The mixture containing the crude compound of Formula I and the non-polarorganic solvent is formed under conditions sufficient to dissolve thecrude compound. The mixture can be heated, if necessary, to dissolve thecompound. Any suitable temperature can be used to dissolve the compoundof Formula I. One of skill in the art will appreciate that the heatingtemperature will depend, in part, on one or more factors including thepolarity of the solvent, the quantity of the solvent, the level ofpurity of the crude compound, and the specific structure of the compoundof Formula I. Such factors will also determine, to an extent, the lengthof time required to dissolve the crude compound. Any suitable length ofthe time can be used, ranging from a few minutes to several hours. Forexample, the mixture containing the crude compound and the organicsolvent can be mixed, with or without heating, for about 10 minutes, orabout 20 minutes, or 30 minutes, or about 40 minutes, or about 1 hour.Accordingly, some embodiments of the invention provide a method forobtaining a purified, solid compound of Formula I as described above,wherein forming the mixture containing the crude compound and theorganic solvent includes heating the mixture. In some embodiments,forming the mixture includes heating the mixture to a temperature offrom about 35° C. to about 100° C. In some embodiments, forming themixture includes heating the mixture to reflux.

In order to separate the purified compound of Formula I from the othercomponents in the crude material, the compound of Formula I issolidified and isolated from the mother liquor of the organic solventmixture. Solidifying the compound of Formula I can include cooling themixture. The mixture can be cooled to any suitable temperature. One ofskill in the art will appreciate that the cooling temperature candepend, in part, on the solubility of the compound of Formula I in theorganic solvent, as well as the quantity of the solvent used in theprocess. In some embodiments, cooling the mixture includes cooling themixture to a temperature of from about −78° C. to about 25° C. In someembodiments, cooling the mixture includes cooling the mixture to atemperature of about 15° C. The cooling can be conducted over anysuitable length of time, typically ranging from a few minutes to severalhours. For example, the mixture can be slowly cooled to a desiredtemperature over a period of three to four hours. After the mixturereaches a desired temperature, it can be held at or around thattemperature for an additional period ranging from a few minutes toseveral hours.

Separating the solidified compound from the organic solvent can beaccomplished by a number of techniques, including passing the mixturethrough a filter to isolate the solid material from the mother liquor orcentrifuging the mixture and removing the mother liquor supernatant. Theseparated solid material can be triturated with additional portions ofthe organic solvent to remove residual impurities, if present. Themother liquor can include varying quantities of the compound of FormulaI that was not solidified, however. If the mother liquor contains suchunsolidified material, the unsolidified material can be recovered, e.g.,by removal of the organic solvent under vacuum, and resubjected to thedissolution/solidification steps. Accordingly, some embodiments of theinvention provide methods as described above, further includingisolating any unsolidified crude compound having a structure accordingto Formula I. In some embodiments, the method further includesconducting steps a), b), and c), as described above, with theunsolidified crude compound.

The purified, solid compound can be obtained in a number of forms usingthe methods of the invention. The compound of Formula I can be obtained,for example, as a powder, a glass, or a foam. The compound of Formula Ican be obtained as a crystalline form or an amorphous form. The compoundcan be obtained as a mixture of two or more forms. In some embodiments,the purified solid compound is obtained in a crystalline form. Acrystalline form is characterized by constituent molecules that arepacked in a regularly ordered, repeating pattern and generally extendingin all three spatial dimensions. In some embodiments, the purified solidcompound is obtained in an amorphous form. An amorphous form is a solidform having no definite crystal structure, i.e., lacking a regularlyordered, repeating pattern of constituent molecules.

One of skill in the art will appreciate that the solid form of thepurified compound will depend to an extent on the structure of thecompound and the characteristics of the solvent used in thedissolution/solidification steps. The purified, solid compound—obtainedin a particular form as described above—can be further processed toobtain a more preferred solid form if necessary. In some embodiments,the method as described above further includes: d) solubilizing thesolidified compound in a polar organic solvent to form a solution; e)contacting the solution with water to precipitate at least a portion ofthe compound; and f) drying the precipitated compound.

Any suitable polar organic solvent can be used to dissolve the purified,solid compound. In general, the most suitable polar organic solvents aremiscible with water. Examples of polar organic solvents include, but arenot limited to, dimethylformamide, dimethyl sulfoxide, propylenecarbonate, acetonitrile, methanol, ethanol, isopropanol, t-butanol,tetrahydrofuran, acetone, and mixtures thereof. Preferred polar organicsolvents have boiling points below 100° C. In some embodiments, thepolar organic solvent is selected from methanol, ethanol, isopropanol,t-butanol, tetrahydrofuran, and acetone. In some embodiments, the polarorganic solvent is methanol. In general, the ratio of polar organicsolvent to the purified, solid compound ranges from about 1:1 by weightto about 500:1 by weight. The ratio of polar organic solvent to thepurified, solid compound can be, for example, about 2:1 by weight orabout 3:1 by weight.

Water is added to the solution of the purified compound of Formula I inthe polar organic solvent, generally in an amount sufficient toprecipitate the compound of Formula I from the solution. Any suitableamount of water can be used in the methods of the invention. Typically,the ratio of water to polar organic solvent ranges from about 1:20 toabout 20:1 by volume. The ratio of water to polar organic solvent canbe, for example, about 10:1. Following precipitation, the precipitatedcompound can be isolated via filtration or centrifugation as describedabove. Alternatively, the mixture can be frozen and the solvent/watermixture can be removed from the precipitate via sublimation. In someembodiments, the precipitated compound is dried under reduced pressure.Any suitable pressure and drying time can be used to remove traces ofwater and solvent from the precipitated compounds. Drying can beconducted, for example, under reduced pressure until the weight of theprecipitated compound remains constant.

The methods of the present invention can be used to purify a number ofmacrolide derivatives. Macrolides, including those having structuresaccording to Formula II in FIG. 2, are polyketide natural products andsynthetic analogs characterized by a macrocyclic lactone ring. Themethods of the invention are particularly useful for preparation ofrapamycin derivatives Biolimus A9 (BA9), everolimus, zotarolimus, andtemsirolimus. Some embodiments of the invention provide methods forobtaining a purified solid compound of Formula I as described above,wherein R¹ is selected from H and R^(a)—(O)_(d)—R^(b) In someembodiments, R¹ is selected from H, CH₂—CH₂—OH, and CH₂—CH₂—O—CH₂—CH₃.In some embodiments, R¹ is CH₂—CH₂—O—CH₂—CH₃. Other macrolidederivatives can also be purified using the methods of the invention.

The methods of the invention provide highly pure compounds of Formula I.In general, the purified, solid compounds are at least 90% pure. In someembodiments, the purified, solid compounds are at least 95% pure. Insome embodiments, the purity of the purified, solid compound isincreased by from about 1% to about 20% with respect to the crudecompound. The methods of the invention can provide greater increases inthe purity of the purified, solid compound depending, in part, onfactors such as the structure of the particular rapamycin derivative andthe starting purity of the crude material. In some embodiments, thepurity of the purified, solid compound is increased by from about 1% toabout 10% with respect to the crude compound. In some embodiments, thepurity of the purified, solid compound is increased by about 3% withrespect to the crude compound.

In some embodiments, the methods of the invention also includecontacting the purified, solid compound with an anti-solventcomposition. The methods of the invention can include combining asolution of the purified compound with an anti-solvent composition. Ingeneral, the anti-solvent composition contains a non-polar organicsolvent in which the compound is insoluble or sparingly soluble at orbelow room temperature. The anti-solvent composition can contain, forexample, diethyl ether, diisopropyl ether, methyl t-butyl ether,ligroin, cyclohexane, octane, hexane, heptane, and mixtures thereof. Insome embodiments, the anti-solvent composition includes diethyl ether,diisopropyl ether, hexane, heptane, or any combination thereof. In someembodiments, the anti-solvent composition includes hexane, heptane,ligroin, octane, cyclohexane, and mixtures thereof. In some embodiments,the methods include contacting the purified compound with ananti-solvent composition and obtaining a crystalline solid form of thecompound.

In a related aspect, the invention provides a crystalline form of40-O-(2-ethoxyethyl) rapamycin. The crystalline form exhibits superiorstability as compared to the amorphous form.

In some embodiments, the crystalline form of 40-O-(2-ethoxyethyl)rapamycin is such that the differential scanning calorimetry thermogramthereof shows one or more local minima at around 138° C. and around 192°C. In some embodiments, the thermogram shows local minima at or around137.9° C. and 191.6° C. In some embodiments, the thermogram issubstantially in accordance with FIG. 5 as shown herein.

In some embodiments, the crystalline form of 40-O-(2-ethoxyethyl)rapamycin is such that the infrared spectrum thereof shows one or morepeaks at or around the wavenumbers selected from 2967.1 cm⁻¹, 2931.7cm⁻¹, 2863.3 cm⁻¹, 1745.9 cm⁻¹, 1718.8 cm⁻¹, 1645.7 cm⁻¹, 1619.0 cm⁻¹,1451.2 cm⁻¹, 1378.2 cm⁻¹, 1189.4 cm⁻¹, 1073.9 cm⁻¹, and 988.1 cm⁻¹. Insome embodiments, the infrared spectrum shows five or more peaks at oraround the wavenumbers selected from 2967.1 cm⁻¹, 2931.7 cm⁻¹, 2863.3cm⁻¹, 1745.9 cm⁻¹, 1718.8 cm⁻¹, 1645.7 cm⁻¹, 1619.0 cm⁻¹, 1451.2 cm⁻¹,1378.2 cm⁻¹, 1189.4 cm⁻¹, 1073.9 cm⁻¹, and 988.1 cm⁻¹. In someembodiments, the infrared spectrum shows ten or more peaks at or aroundthe wavenumbers selected from 2967.1 cm⁻¹, 2931.7 cm⁻¹, 2863.3 cm⁻¹,1745.9 cm⁻¹, 1718.8 cm⁻¹, 1645.7 cm⁻¹, 1619.0 cm⁻¹, 1451.2 cm⁻¹, 1378.2cm⁻¹, 1189.4 cm⁻¹, 1073.9 cm⁻¹, and 988.1 cm⁻¹. In some embodiments, theinfrared spectrum is substantially in accordance with FIG. 12 as shownherein.

In some embodiments, the crystalline form of 40-O-(2-ethoxyethyl)rapamycin is such that the X-ray powder diffraction pattern thereofshows one or more diffraction peaks at or around the angles (2θ)selected from the group consisting of 5.00°, 7.06°, 9.22°, 10.07°,10.50°, 11.94°, 12.71°, 13.15°, 14.73°, 16.33°, 16.80°, 17.07°, 18.01°,18.57°, 19.42°, 19.81°, 20.16°, 20.44°, 20.93°, 21.55°, 22.29°, 22.58°,23.92°, 24.26°, 24.83°, 25.17°, 26.32°, 27.48°, 28.60°, and 32.28°. Insome of these embodiments, the X-ray powder diffraction pattern thereofshows 10 or more diffraction peaks at or around the angles (2θ) selectedfrom the group consisting of 5.00°, 7.06°, 9.22°, 10.07°, 10.50°,11.94°, 12.71°, 13.15°, 14.73°, 16.33°, 16.80°, 17.07°, 18.01°, 18.57°,19.42°, 19.81°, 20.16°, 20.44°, 20.93°, 21.55°, 22.29°, 22.58°, 23.92°,24.26°, 24.83°, 25.17°, 26.32°, 27.48°, 28.60°, and 32.28°. In stillsome of these embodiments, the X-ray powder diffraction pattern thereofshows 25 or more diffraction peaks at or around the angles (2θ) selectedfrom the group consisting of 5.00°, 7.06°, 9.22°, 10.07°, 10.50°,11.94°, 12.71°, 13.15°, 14.73°, 16.33°, 16.80°, 17.07°, 18.01°, 18.57°,19.42°, 19.81°, 20.16°, 20.44°, 20.93°, 21.55°, 22.29°, 22.58°, 23.92°,24.26°, 24.83°, 25.17°, 26.32°, 27.48°, 28.60°, and 32.28°. In someembodiments, the X-ray powder diffraction pattern shows one or morediffraction peaks at or around the angles (2θ) shown in Table 4. In someembodiments, the X-ray powder diffraction pattern shows 10 or more, or25 or more, diffraction peaks at or around the angles (2θ) shown inTable 4. In some embodiments, the X-ray powder diffraction pattern issubstantially in accordance with FIG. 6 as shown herein.

IV. Examples Example 1 Purification of BA9

Following synthesis of BA9 according to FIG. 2, a chromatographygradient containing n-hexane and ethyl acetate in various ratios wasused as a first purification step. Ninety fractions were collectedduring chromatography. Fractions 15-30 were combined to form Lot A(containing 3.6 g of BA9). Fractions 31-50 were combined to form Lot B(containing 4.6 g of BA9). Fractions 51-70 were combined to form Lot C(containing 3.4 g of BA9). Fractions 1-14 and 71-90 were combined toform Lot E (containing 2.2 g of BA9). The various lots contained92.0-94.9% BA9, as determined by HPLC and summarized in Table 1 below.Relative amounts are expressed as AUC % (area under the curve), i.e.,the fraction of the total signal that corresponds to a single peak(corresponding, in turn, to a single species, or two or more specieswith similar/identical absorbance wavelengths and retention times) in agiven chromatogram. Impurities 1-9 shown in Table 1 are numberedaccording to increasing retention time; retention times werereproducible across experiments.

TABLE 1 Lots of purified BA9 from the second column chromatographyRelative Quantity (% AUC, as determined by HPLC) of BA9 and Impurities1-9 Lot 1 2 3 BA9 4 5 6 7 8 9 A <0.1 0.09 0.58 91.5 0.15 0.80 0.82 3.740.47 1.40 B <0.1 0.08 0.89 92.8 0.27 0.79 0.63 3.11 0.37 0.69 C 0.340.10 1.16 88.7 1.20 1.36 0.84 4.91 0.82 0.43 D <0.1 <0.1 <0.1 85.1 1.161.67 1.17 6.32 1.16 3.37 E <0.39 0.14 0.83 76.0 3.46 2.31 2.12 8.07 1.513.93

To Lot B, a viscous crude material containing 4.6 g of BA9, was addedn-hexane (490 g) at room temperature. While the mixture was vigorouslystirred, it was heated to reflux (˜70° C.) within 30 minutes. Theviscous material turned to an easily-stirred, suspended powder at ˜56°C. and then to a clear solution after ˜5 minutes stirring at reflux. Thesystem was refluxed for 20 minutes and then allowed to cool to roomtemperature within 3 hours. The cooling caused precipitation of a whitesolid. The suspension was additionally stirred at 25-15° C. for 2 hours.The suspension was then filtered, and the isolated solid was washed onthe filter with 50 g of n-hexane. The solid was then dried in vacuo to aconstant weight (yield=3.4 g, 74% recovery of BA9).

The results of purification are summarized in Table 2. The precipitationprocedure lowered the amounts of the late-eluting BA9 impurities (i.e.,impurities 5-9). The amount of impurity 4, as well as of other morepolar impurities, were similar for the starting lot and thepurified/precipitated lot of BA9.

TABLE 2 HPLC analysis of BA9 Lot B (refer to Table 1) purified byprecipitation from hot n-hexane Relative Quantity (% AUC, as determinedby HPLC) of BA9 and Impurities 1-9 Portion 1 2 3 BA9 4 5 6 7 8 9Starting Lot <0.1 0.08 0.89 92.8 0.27 0.79 0.63 3.11 0.37 0.69 (Lot B)Purified, solid 0.12 0.09 0.87 96.0 0.32 0.32 0.32 1.79 <0.1 0.19 BA9(Lot B) Mother liquor <0.1 0.21 0.63 68.4 <0.1 4.36 4.11 12.00 2.75 3.39after purification

Lots A, C and E were also precipitated from refluxing n-hexane. Forthis, a similar weight ratio of 1:110 between the weight of BA9(established by quantitative HPLC analysis) and n-hexane was used. Theamounts and purities of the four lots of BA9 obtained by the n-hexanepurification are summarized in Table 3. As it is evident from Table 3,the purification of portions of BA9 by precipitation from refluxingn-hexane generated two new lots A and B (presented in Table 3)containing BA9 with improved levels of purity (i.e., >95%). The lotswere obtained using the surprisingly simple, economical method of theinvention. Table 3 shows improved values related to Table 1.

TABLE 3 HPLC Analysis of portions of purified BA9 by precipitation fromrefluxing n-hexane - refer to corresponding portions from Table 1.Relative Quantity (% AUC, as determined by HPLC) of BA9 and Impurities1-9 Lot 1 2 3 BA9 4 5 6 7 8 9 Weight A¹ <0.1 0.07 0.59 96.5 0.16 0.250.27 1.46 0.06 0.41 2.9 g B¹ 0.12 0.09 0.87 96.0 0.32 0.32 0.32 1.79<0.1 0.19 3.4 g C¹ 0.56 <0.1 1.06 94.0 1.06 0.30 1.03 1.92 <0.1 <0.1 1.6g E¹ 0.68 0.17 1.12 92.6 2.26 0.21 0.49 2.02 <0.1 0.27 1.2 g ¹Product ofn-hexane precipitation.

The discovery that a simple solidification by lowering temperature wouldseparate the 40-O derivatives so cleanly from unwantedimpurities/byproducts was surprising and unpredictable. It iseconomically advantageous because the unwanted by-products are often ofa similar polarity to the desired product and require labor-intensive,high-cost large scale chromatography techniques for removal.

Example 2 Preparation and Characterization of BA9 Solid Forms

Batch Profiling and Polymorph Screening Methods.

BA9 Lots 2A and 2B were purified using precipitation from n-hexanes asdescribed above. After the purification process, Lot 2A (82.9 g) wasdissolved in methanol (190 g) and precipitated by adding the methanolsolution to water. The combined methanolic solution was transferred toan addition funnel and then slowly charged within 1 hour and 15 minutesinto vigorously stirred water for injection (2.18 kg) in a reactor at atemperature range of 0-5° C. After completion of the addition of themethanolic solution of BA9, the resulting white suspension was stirredfor an additional 15 minutes at the same temperature range. Theprecipitated BA9 was isolated in 84.8% yield (purity was 96.3% asdetermined by HPLC). The suspension was then filtered, and the isolatedwhite solid was washed on the filter. Lot 2A was characterized by XRPDand DSC and Lot 2B was characterized by XRPD, DSC, optical microscopy,and infrared (IR) spectroscopy. Lot 2A was found to be amorphous, asassessed by XRPD. Lot 2B was found to be crystalline, as assessed byXRPD, and the crystalline form was designated as Form I. A dynamic vaporsorption (DVS) experiment was run on Lot 2B.

Two stock solutions of BA9 Lot 2A in EtOAc and acetone were prepared.These solutions were dispensed into glass vials and placed in a vacuumoven for evaporation to dryness. The solids obtained after drying weredissolved in different solvents/solvent mixtures and stirred overnight.Any solids obtained after the dissolution-recrystallization process wereanalyzed as wet cake by XRPD.

Anti-solvent crystallization experiments were carried out by dissolvingLot 2A in 6 different organic solvents. Hexane was added as theanti-solvent. If no solids were observed, the solutions were allowed tostir at room temperature.

Results and Discussion

Batch Profiling.

BA9 Lot 2A was characterized by XRPD (FIG. 3) and DSC (FIG. 4) and Lot2B was characterized by XRPD (FIG. 5), DSC (FIG. 6), optical microscopy(FIG. 7), and infrared spectroscopy (FIG. 12). Lot 2A is amorphous byXRPD. Lot 2B is crystalline by XRPD and was designated as Form I.Diffraction angles (2θ) for Form I are listed in Table 4.

TABLE 4 BA9 Form I XRPD Data Angle, 2θ d spacing Intensity, % 5.00 17.6667.7 7.06 12.51 6.6 9.22 9.58 5.7 10.07 8.77 28.8 10.50 8.42 67.6 11.947.40 3.8 12.71 6.96 31.2 13.15 6.73 18.3 14.73 6.01 100 16.33 5.42 34.516.80 5.27 25.1 17.07 5.19 39.8 18.01 4.92 27.5 18.57 4.77 16.3 19.424.57 4.3 19.81 4.48 11.6 20.16 4.4 26.4 20.44 4.34 8.9 20.93 4.24 21.421.55 4.12 18.5 22.29 3.99 4.9 22.58 3.93 4.6 23.92 3.72 15.9 24.26 3.6710.1 24.83 3.58 2.4 25.17 3.54 9.4 26.32 3.38 10.8 27.48 3.24 6 28.603.12 4.7 32.28 2.77 2.4

A DVS experiment was run on Lot 2B. The increase in mass of the samplewas ˜0.5% at 90% RH. The kinetic plot is shown in FIG. 8. The post DVSsample was analyzed by XRPD and confirmed as Form I. FIG. 9 shows anoverlay of the XRPD patterns of the post-DVS sample and a referencepattern of Form I.

Polymorph Screening.

Two stock solutions of BA9 Lot 2A (100 mg/ml) in ethyl acetate (Stock Ain Table 4) and acetone (Stock B in Table 4) were prepared by dissolving˜380 mg of the material in 3.8 ml of the respective solvents. Thesesolutions were dispensed into 22 glass vials (0.4 ml in each vial, 11vials per stock solution) and placed in a vacuum oven at roomtemperature for evaporation to dryness. After 2 days of drying ˜40 mgmaterial were obtained in each vial as a cracked gel.

Eleven different solvents/solvent mixtures were added to the vials (160μl each) and the solutions obtained were allowed to stir at roomtemperature. After overnight stirring, if solids had precipitated out,then the slurry was filtered and the solids obtained were analyzed aswet cake by XRPD. The results of these experiments are shown in Table 5.FIG. 10 shows an overlay of the XRPD patterns obtained in therecrystallization experiments and a reference pattern of Form I.

TABLE 5 Summary of Dissolution/Recrystallization Experiments Sample No.Solvent Stock A Stock B 3 DEE Form I Form I 4 DIPE Form I Form I 5 MTBEClear Solution Clear Solution 6 Hexane Form I Form I 7 Heptane Form IForm I 8 MEK Clear Solution Clear Solution 9 Toluene Clear SolutionClear Solution 10 IPA:Water 1:1 N/A (gel N/A (gel precipitate)precipitate) 11 Acetone:Water 1:1 N/A (gel N/A (gel precipitate)precipitate)

Anti-solvent crystallization experiments were carried out by dissolving˜40 mg each of Lot 2A in 6 different organic solvents (200 0 each).Hexane was added as the antisolvent in steps of 200 μl until solidsprecipitated out or 1.6 ml of hexane had been added. If no solids wereobserved the solutions were allowed to stir at room temperature. Theresults of these experiments are shown in Table 6. FIG. 11 shows anoverlay of the XRPD patterns obtained in the anti-solventcrystallization experiments and a reference pattern of Form I.

TABLE 6 Summary of Anti-Solvent Crystallization Experiments. Vol. Formas Sample of Hexane determined by No. Solvent Added XRPD Comment 12Ethyl 1.6 mL Form I Precipitated after a few Acetate min. of stirring 13Acetone 1.6 mL N/A No solids 14 IPA 1.6 mL Form I Precipitated after30-45 min. of stirring 15 EtOH 1.6 mL N/A No solids 16 THF 1.6 mL Form IPrecipitated after >1 hr. of stirring 17 DEE 400 μL  Form I Gel-likeprecipitate changed to white solid after few min. of stirring

Example 3 Crystalline BA9 Exhibits Superior Stability

Photolysis studies were performed on amorphous and crystalline BA9 (i.e,Form I). After exposure to a minimum of 1.2 million lux hours and notless than 200 W·hours/m² per ICH Q1B, both the crystalline and amorphousBA9 exhibited significant increases in degradation compounds withsimilar retention times. However, photolysis of crystalline BA9 resultedin less overall major degradants as determined by HPLC, refer to Table7. Furthermore, purity assay measurements showed that after photolysis,the crystalline BA9 assayed at 70.2% pure, which was substantiallyhigher than the 16.3% assay purity of the amorphous BA9.

The improvement in stability of the crystalline BA9 as compared to theamorphous BA9 is further supported by the comparison of the DarkControls, which were maintained at standard laboratory temperature andsame conditions as the test samples with the exception of exposure tolight for the duration of the study. The crystalline Dark Control (assay98.0%) also exhibited improved stability when compared with theamorphous Dark Control (88.2%). Therefore, the crystalline BA9 was shownto be significantly more stable than amorphous BA9 under photolysis andstorage under laboratory conditions.

TABLE 7 Summary of Photostability Experiments Crystalline BA9 AmorphousBA9 T = 0 Photolysis - Dark T = 0 Photolysis - Dark Control VIS/UVControl Control VIS/UV Control Purity Assay, % 97.7 70.2  98.0  98.016.3  88.2  RT (min) Major Degradants, % Major Degradants, % 4.2 0.760.01 1.30 — 4.5 2.00 0.08 7.40 0.72 4.9 2.60 0.41 1.20 0.31 6.2 0.940.09 2.50 0.20 7.9 0.07 — 2.20 — 10.4 0.15 0.03 1.70 0.44 10.7 0.69 0.060.94 1.40 11.0 0.51 0.67 2.60 0.39

Oxidation studies were performed on amorphous and crystalline BA9. Afterexposure to 30% H₂O₂ for 3 hours, both the crystalline and amorphous BA9exhibited increases in degradation compounds with similar HPLC retentiontimes. However, oxidative degradation of crystalline BA9 resulted inlower levels of major degradants overall. In addition, assaymeasurements showed that after oxidative degradation, the crystallineBA9 exhibited a weight loss of 3.4% (relative to a control sample),which was substantially lower than the weight loss of 6.5% exhibited byamorphous BA9. It is therefore apparent that amorphous BA9 is moresusceptible to oxidative degradation.

The superior photostability and chemical stability of crystalline BA9can prevent loss of valuable material during storage and fabrication ofcoated medical devices. The advantages of the new crystalline form, aswell as the economy and surprising simplicity of the methods forpreparation of the crystalline form, help to maintain the integrity ofthe drug-coated products while decreasing the cost and complexity oftheir production.

Although the foregoing has been described in some detail by way ofillustration and example for purposes of clarity and understanding, oneof skill in the art will appreciate that certain changes andmodifications can be practiced within the scope of the appended claims.In addition, each reference provided herein is incorporated by referencein its entirety to the same extent as if each reference was individuallyincorporated by reference.

What is claimed is:
 1. A method for obtaining a purified, solid compoundhaving a structure according to Formula I:

wherein R¹ is selected from the group consisting of R^(a)—(O)_(d)—R^(b),wherein R^(a) is C₁₋₅alkylene, R^(b) is C₁₋₅alkyl or C₁₋₅alkylene-OH,and the subscript d is an integer selected from 0-1; C₁₋₅alkyl;C₆₋₁₀arylC₁₋₅alkyl; hydroxyC₁₋₅alkyl; C₆₋₁₀arylC₁₋₅alkoxy;C₁₋₅alkoxyC₁₋₅alkyl; acyl; acylC₁₋₅alkyl; aminoC₁₋₅alkyl;C₁₋₅alkylaminoC₁₋₅alkyl; acylaminoC₁₋₅alkyl;C₁₋₅alkoxycarbonylaminoC₁₋₅alkyl; and C₆₋₁₀aryl; the method comprising:a) forming a mixture consisting essentially of a crude compound having astructure according to Formula I and a non-polar organic solvent underconditions sufficient to dissolve the compound, wherein the dielectricconstant of the non-polar organic solvent is less than about 5; b)solidifying at least a portion of the compound having the structureaccording to Formula I; and c) separating at least a portion of thesolidified compound from the solvent in the mixture; thereby obtainingthe purified, solid compound.
 2. The method of claim 1, wherein thenon-polar organic solvent is selected from the group consisting ofhexane, heptane, methyl t-butyl ether, ligroin, octane, cyclohexane, andmixtures thereof.
 3. The method of claim 2, wherein the non-polarorganic solvent is hexane.
 4. The method of claim 2, wherein thenon-polar organic solvent is heptane.
 5. The method of claim 1, whereinforming the mixture in step a) comprises heating the mixture.
 6. Themethod of claim 5, wherein forming the mixture in step a) comprisesheating the mixture to a temperature of from about 35° C. to about 100°C.
 7. The method of claim 5, wherein forming the mixture in step a)comprises heating the mixture to reflux.
 8. The method of claim 1,wherein solidifying a portion of the compound in step b) comprisescooling the mixture.
 9. The method of claim 8, wherein cooling themixture in step b) comprises cooling the mixture to a temperature offrom about −78° C. to about 25° C.
 10. The method of claim 9, whereincooling the mixture in step b) comprises cooling the mixture to atemperature of about 15° C.
 11. The method of claim 1, wherein thepurified, solid compound is obtained in a crystalline form.
 12. Themethod of claim 1, wherein the purified, solid compound is obtained inan amorphous form.
 13. The method of claim 1, further comprising: d)solubilizing the solidified compound in a polar organic solvent to forma solution; e) contacting the solution with water to precipitate atleast a portion of the compound; and f) drying the precipitatedcompound.
 14. The method of claim 13, wherein the polar organic solventis selected from the group consisting of methanol, ethanol, isopropanol,t-butanol, tetrahydrofuran, and acetone.
 15. The method of claim 14,wherein the polar organic solvent is methanol.
 16. The method of claim13, wherein the drying is conducted under reduced pressure.
 17. Themethod of claim 1, further comprising: i) contacting the compound withan anti-solvent composition; and ii) obtaining a crystalline solid formof the compound.
 18. The method of claim 13, further comprising: i)contacting the compound with an anti-solvent composition; and ii)obtaining a crystalline solid form of the compound.
 19. The method ofclaim 17, wherein the compound is purified and solid.
 20. The method ofclaim 17, wherein the compound is purified.
 21. The method of claim 17,wherein the compound is solid.
 22. The method of claim 17, wherein theanti-solvent composition comprises hexane, heptane, methyl t-butylether, ligroin, octane, cyclohexane, and mixtures thereof.
 23. Themethod of claim 1, wherein R¹ is R^(a)—(O)_(d)—R^(b).
 24. The method ofclaim 23, wherein R¹ is selected from the group consisting of CH₂—CH₂—OHand CH₂—CH₂—O—CH₂—CH₃.
 25. The method of claim 24, wherein R¹ isCH₂—CH₂—O—CH₂—CH₃.
 26. The method of claim 1, wherein the purity of thepurified, solid compound is increased by from about 1% to about 20% withrespect to the crude compound.
 27. The method of claim 1, wherein thepurity of the purified, solid compound is increased by from about 1% toabout 10% with respect to the crude compound.
 28. The method of claim 1,wherein the purity of the purified, solid compound is increased by about3% with respect to the crude compound.
 29. A crystalline form of40-O-(2-ethoxyethyl) rapamycin.
 30. The crystalline form of claim 29,wherein the X-ray powder diffraction pattern thereof shows one or morediffraction peaks at or around the angles (2θ) selected from the groupconsisting of 5.00°, 7.06°, 9.22°, 10.07°, 10.50°, 11.94°, 12.71°,13.15°, 14.73°, 16.33°, 16.80°, 17.07°, 18.01°, 18.57°, 19.42°, 19.81°,20.16°, 20.44°, 20.93°, 21.55°, 22.29°, 22.58°, 23.92°, 24.26°, 24.83°,25.17°, 26.32°, 27.48°, 28.60°, and 32.28°.
 31. The crystalline form ofclaim 29, wherein the X-ray powder diffraction pattern thereof shows 10or more diffraction peaks at or around the angles (2θ) selected from thegroup consisting of 5.00°, 7.06°, 9.22°, 10.07°, 10.50°, 11.94°, 12.71°,13.15°, 14.73°, 16.33°, 16.80°, 17.07°, 18.01°, 18.57°, 19.42°, 19.81°,20.16°, 20.44°, 20.93°, 21.55°, 22.29°, 22.58°, 23.92°, 24.26°, 24.83°,25.17°, 26.32°, 27.48°, 28.60°, and 32.28°.
 32. The crystalline form ofclaim 29, wherein the X-ray powder diffraction pattern thereof shows 25or more diffraction peaks at or around the angles (2θ) selected from thegroup consisting of 5.00°, 7.06°, 9.22°, 10.07°, 10.50°, 11.94°, 12.71°,13.15°, 14.73°, 16.33°, 16.80°, 17.07°, 18.01°, 18.57°, 19.42°, 19.81°,20.16°, 20.44°, 20.93°, 21.55°, 22.29°, 22.58°, 23.92°, 24.26°, 24.83°,25.17°, 26.32°, 27.48°, 28.60°, and 32.28°.